Original Article Protective effects of curcumin on hepatocytes in cecal ligation and puncture-induced sepsis in rats

Original Article

Protective effects of curcumin on hepatocytes in cecal

ligation and puncture-induced sepsis in rats

Hai-Yan Yin1_{, Pei Tao}1_{, Jian-Rui Wei}2_{, Rui Zhang}1_{, Yong-Hui Ma}1

1_{Department of Intensive Care Unit, Guangzhou Red Cross Hospital, Medical College, Jinan University, Guangzhou} 510220, PR China; 2_{Department of Cardiology, Guangzhou Red Cross Hospital, Medical College, Jinan University,} Guangzhou 510220, PR China

Received November 14, 2015; Accepted August 13, 2016; Epub December 15, 2016; Published December 30, 2016

Abstract: We aim to observe the protective effects of curcumin in hepatocytes in septic rat. Sprague-Dawley rats were randomly divided into six groups of 20 rats each: sham group; model group; xuebijing group; low dose group (50 mg/kg), middle dose group (100 mg/kg) and high dose group (150 mg/kg). Another 60 rats were randomly divided into the six above-mentioned experimental groups of 10 rats each to determine the survival rate. Our results showed that, treatment with curcumin and Xuebijing significantly attenuated the CLP-induced hepatocyte edema and inflammation indicators, especially in the Xuebijing and 100-mg/kg curcumin groups. This result was also sup -ported by the histopathologic examination findings. Additionally, curcumin improved the survival rate of rats with CLP. Curcumin has a protective effect on CLP-induced liver dysfunction and that the effect varies at different doses; the 100-mg/kg dose was optimal for protection of hepatocytes in septic rats. This protective effect can be attributed to the ability of curcumin to counteract inflammatory cell infiltration and regulate cytokines.

Keywords: Curcumin, sepsis, hepatocyte, cecal ligation, puncture

Introduction

Sepsis is a systemic, deleterious host response to infection and a common complication of burns, trauma, shock, and major surgery. It may lead to septic shock, multiple organ dysfunc-tion syndrome, and other serious condidysfunc-tions. The mortality rate associated with sepsis is extremely high; the overall hospital mortality rate is reportedly 28.6%, while the mortality rate associated with severe sepsis and septic shock are higher at 25% to 30% and 40% to 70%, respectively [1, 2]. Sepsis has become the leading cause of death in the intensive care unit, causing more deaths than acute myocar-dial infarction, lung cancer, and breast cancer and killing 1,400 people worldwide each day [3-5]. Moreover, the cost associated with sep-sis is remarkably high. According to an epide-miological survey, more than 370,000 people die of sepsis in Europe and the United States each year, and the cost of treatment reach up to $25 billion [6]. Sepsis has thus become a serious problem and challenge to human health [7]. The development of effective treatments

and drugs for sepsis is significant and

emergent.

As the central organ of metabolism, the liver is one of the most important organs participating in the elimination of bacteria and endotoxins from the body [8-10]. Numerous studies have

identified a close relationship between the

development of liver injury and the occurrence and aggravation of sepsis and septic shock

[11]. Liver dysfunction plays a critical role in the

disease course [9]. Patients with sepsis have

significantly high levels of oxygen free radicals and release of inflammatory mediators, which

occurs secondary to increased membrane per-meability caused by damage to the liver cell structures and mitochondrial membranes. Sodium pump dysfunction leads to sodium

retention, hepatocyte swelling, and finally hepa -tocyte apoptosis [12-15].

anti-oxi-dation, and anti-inflammation effects [16].

Curcumin also exhibits relatively low toxicity, has a simple extraction process, and is low in

cost. Curcumin has been confirmed to be

extremely safe in various animal models and clinical studies, even at the maximum daily dose of 12 g, and can be administered topical-ly, oraltopical-ly, and by inhalation. Therefore, curcumin shows great potential for clinical applications [17]. Curcumin has a dual role in apoptosis, showing different reactions in different cells. Previous studies have revealed that curcumin exerts a protective effect against liver cell inju-ry caused by multiple factors including ethanol-induced and carbon tetrachloride-ethanol-induced liver cell damage [18-20]. The underlying mecha-nism involves increased superoxide dismutase

vitality, regulation of inflammatory and anti-inflammatory cytokine expression, inhibition of

cytochrome C activity, and other processes. Although curcumin has been shown to be

ben-eficial in sepsis, its potential value in protection

of sepsis-induced hepatocyte dysfunction has not been evaluated [20, 21].

Therefore, the present study was designed to demonstrate the protective effect of curcumin and its possible mechanism on hepatocytes in septic rats. We also explored the potential clini-cal use of curcumin for treatment of sepsis-induced liver dysfunction.

Materials and methods

Experimental animals

Male 3-month-old specific-pathogen-free

Spra-gue-Dawley rats weighing 250 to 350 g were

obtained from the Laboratory Animal Center of

Sun Yat-sen University (Guangzhou, China). The rats were maintained in cages in a tempera-ture-controlled room at 25°C±1°C, under an

artificial 12-h light-dark cycle, and on a stan -dard diet and water. Animal housing and all experimental procedures were approved by the

Institute of Laboratory Animal Science, Jinan

University.

Chemicals and reagents

Curcumin of analytical grade was purchased

from Sigma-Aldrich (St. Louis, MO, USA).

Xuebijing (XBJ) (batch number 1308221) was supplied by Tianjin Chase Sun Pharmaceutical

Co., Ltd. (Tianjin, China). All other chemicals

and biochemicals used in this study were of high analytical grade.

Animal groups

Sprague-Dawley rats were randomly divided into six groups of 20 rats each: animals

under-going sham CLP (Sham group), animals under

-going CLP (CLP group), animals under-going CLP

and treated with XBJ (XBJ group), and animals

undergoing CLP and treated with curcumin at 50 mg/kg (low-dose curcumin [L-Cur] group),

100 mg/kg (middle-dose curcumin [M-Cur] group), or 150 mg/kg (high-dose curcumin [H-Cur] group). Curcumin was diluted with nor-mal saline solution to 10 ml/kg and freshly pre-pared on the day of the experiment. It was

administered intraperitoneally after CLP. The

rats received an additional dose of curcumin at

8, 16, and 24 h post-CLP.

Another 60 experimental rates were observed to determine the survival rate. The rats were randomly divided into the six above-described experimental groups of 10 rats each. XBJ and curcumin were intraperitoneally injected three times a day beginning after surgery and con-tinuing for 3 days. The survival rates were recorded at 12, 24, 36, 48, and 72 h after surgery.

Establishment of animal model

The rat models of sepsis in this study were

layer by layer [22]. After the surgery, sterile

nor-mal saline solution (100 mL/kg of body weight) was administered for fluid resuscitation.

In the Sham group, the abdominal cavity was

opened and the cecum was flipped over and

placed back into the abdominal cavity without ligation or puncture; the remaining steps were the same.

Specimen collection

The animals were anesthetized with 2% sodium

pentobarbital at 2, 6, 12, and 24 h post-CLP (n

= 5 per time point), and the abdominal cavity was opened to collect liver tissue specimens and portal venous and peripheral venous blood samples. Some samples were stored at -80°C until analysis.

Histological evaluation

The harvested liver specimens were fixed in

10% paraformaldehyde for 24 h, dehydrated in

a graded alcohol series, embedded in paraffin,

and serially sectioned. Some of the specimens were stained with hematoxylin and eosin to assess morphological changes. The remaining specimens underwent terminal deoxynucleoti-dyl transferase-mediated dUTP nick end

label-ing (TUNEL) stainlabel-ing uslabel-ing an In Situ Cell Death Detection Kit (Hoffmann-La Roche, Ltd., Basel,

Switzerland). The occurrence of tissue damage was evaluated independently by a pathologist and histologist blinded to the experiment.

Serological evaluation

The serum concentrations of procalcitonin

(PCT), tumor necrosis factor-α (TNF-α), and interleukin-1β (IL-1β) were analyzed by

enzyme-linked immunosorbent assay (Boster Biological

Engineering Co., Ltd., Wuhan, China). The plas -ma samples were centrifuged to separate the serum. All experimental procedures were car-ried out according to the manufacturer’s instructions.

Statistical analysis

All experimental data were statistically pro-cessed using SPSS 13.0 software (SPSS, Inc.,

Chicago, IL, USA). Measurement data are

expressed as mean ± standard error, and a

t-test was performed. Multiple comparisons

were analyzed for significant differences using

one-way analysis of variance with Tukey’s post hoc test for multiple comparisons. A P level of <

0.05 was considered statistically significant.

Results

Histopathologic findings (Figure 1)

In the Sham group, the liver tissues exhibited normal histological features at all postopera-tive time points, including normal hepatocytes, portal areas, and parenchyma.

observed. The central vein and hepatic sinus were conspicuous, sinusoids were mildly dilat-ed, and ballooning degeneration of

hepatocy-tes was partly visible among periportal inflam

-matory cell infiltration. The XBJ and curcumin

groups also showed varying degrees of histo-logical changes, central vein and hepatic sinus congestion, and sinusoidal dilation. Neverthe-

less, this congestion and dilation were signifi

-cantly less severe than those in the CLP group. Six hours after surgery in the CLP group, central

vein and hepatic sinus congestion and sinusoi-dal dilation were widespread. Hepatocyte

hypertrophy and infiltration of inflammatory

cells were more visible. A small amount of liver cell degeneration and punctate eosinophilic necrosis could be seen at this time point. In the XBJ and curcumin groups, the degrees of cen-tral vein and hepatic sinus congestion and sinu-soidal dilation were aggravated, but still less

severe than those in the CLP group.

Twelve hours after surgery, the range of dam-age continued to expand in all groups. Hepa- tocytes showed varying degrees of irregular

arrangements. Fragmentation of hepatic cell

cords and multiple spotty hepatocyte necroses

were widespread in the CLP group. Lesions in the XBJ and curcumin groups were still signifi

-cantly less severe than those in the CLP group.

Twenty-four hours after surgery in the CLP

group, the injury was further aggravated, show-ing a large area of hepatocyte edema and necrosis, accumulation of swollen hepatocytes,

and inflammatory cell infiltration. No worsening

was noted in the XBJ group or curcumin groups.

TUNEL results

Stained apoptotic nuclei appeared as blue-black granule deposits [23]. The apoptotic index was calculated with a formula, the ratio between the number of aptptotic cells and the

Table 1. The changes of apototic index (%) in different time phase

Group Different Time Phase

2 H 6 H 12 H 24 H

A 2.02±0.13 2.10±0.13 2.20±0.05 2.14±0.06

B 23.59±2.00* _30.92±1.69* _50.18±2.11* _52.05±1.31*

C 11.89±1.34*,# _17.76±1.73*,# _27.08±1.64*,# _28.95±1.40*,#

D 17.59±1.43*,#,▲ _23.07±1.18*,#,▲ _33.76±1.58*,#,▲ _34.89±1.76*,#,▲

E 11.56±0.96*,# _18.36±1.10*,# _28.25±1.20*,# _30.35±1.20*,#

F 17.48±1.94*,#,▲ _23.45±2.01*,#,▲ _34.25±1.41*,#,▲ _35.92±1.79*,#,▲

A: Sham group. B: CLP group. C: XBJ group. D: L-Cur group. E: M-Cur group. F: H-Cur group. L-Cur, low-dose curcumin, 50 mg/kg i.p.; M-Cur, middle-dose curcumin, 100 mg/kg i.p.; H-Cur, high-dose curcumin, 150 mg/kg i.p. Data are presented as the mean ± standard error (n = 5). *_{P < 0.05 vs. Sham group;} #_{P < 0.05 vs. CLP group;} ▲_{P < 0.05 vs. XBJ group.}

total number of cells analyzed multiplied by

100 (Table 1). In the Sham group, few to no lower than those in the L-Cur and H-Cur groups (P < 0.05) (Figures 4, 5). Figure 3. Effects of curcumin on the apoptotic index at 6, 12, and 24 h after

surgery. A: Sham group. B: CLP group. C: XBJ group. D: L-Cur group. E: M-Cur group. F: H-M-Cur group. L-M-Cur, low-dose curcumin, 50 mg/kg i.p.; M-M-Cur, middle-dose curcumin, 100 mg/kg i.p.; H-Cur, high-dose curcumin, 150 mg/ kg i.p. Data are presented as the mean ± standard error (n = 5). *P < 0.05 vs. Sham group; #P < 0.05 vs. CLP group; ▲P < 0.05 vs. XBJ group.

Figure 4. Effects of curcumin on PCT level in peripheral blood at 6, 12, and 24 h after surgery. A: Sham group. B: CLP group. C: XBJ group. D: L-Cur group. E: M-Cur group. F: H-Cur group. L-Cur, low-dose curcumin, 50 mg/ kg i.p.; M-Cur, middle-dose curcumin, 100 mg/kg i.p.; H-Cur, high-dose cur-cumin, 150 mg/kg i.p. Data are presented as the mean ± standard error (n = 5). *P < 0.05 vs. Sham group; #P < 0.05 vs. CLP group; ▲P < 0.05 vs. XBJ group.

apoptotic cells were found at each time point. More apop-totic cells were observed in the other groups, with varying de-grees of cytoplasm concentra-tion and karyopyknosis (Figure 2).

Precise information was re-vealed by quantitative analy-sis. The apoptotic index was

significantly higher in the CLP

group than in the XBJ and cur-cumin groups (P < 0.05). The index continued to rise with

time in the CLP group (P < 0.05), while the upward trend ended between the 12- and 24-h time point in the XBJ and curcumin groups (P > 0.05). Additionally, the index was not

significantly different between

the XBJ and M-curcumin groups (P > 0.05), whereas it

was significantly lower than in the L-curcumin and H-curcumin

groups (P < 0.05) (Figure 3).

Serological evaluation

The PCT, TNF-α, and IL-1β lev -els were elevated in all groups

from 2 h post-CLP and were significantly higher in all groups

than in the Sham group (P < 0.05); the most obvious

in-crease was in the CLP group (P

< 0.05). The PCT, TNF-α, and IL-1β levels changed only

sli-ghtly at each time point in the Sham group (P > 0.05), while they continued to rise in the

CLP group (P < 0.05). The lev-els of each parameter peaked

at 12 h post-CLP in the XBJ

group and curcumin groups

and were significantly lower than those in the CLP group (P

< 0.05). Additionally, the differ-ence between the XBJ and M-Cur groups was not

statisti-cally significant (P > 0.05), but

Survival study results

The rats in the CLP group of the survival study

exhibited a 72-h survival rate of only 20%. Conversely, only rats in the XBJ group and cur-cumin groups showed 30% to 50% mortality,

which was significantly lower than that in the CLP group (P < 0.05). All rats in the sham group survived to the 72-h time point (Figure 6).

easily cause abdominal infection. This disease process is similar to that of peritonitis second-ary to perforating appendicitis in clinical prac-tice. Studies have shown that the animal exhib-its a high-power cycle and metabolic state in the early stage, and then enters a low-power cycle later. Additionally, compared with other animal models both the endotoxin detection rate in peripheral blood and bacterial culture-Figure 5. Effects of curcumin on cytokine production in peripheral blood at 6, 12, and 24 h after surgery. A: Sham group. B: CLP group. C: XBJ group. D: L-Cur group. E: M-Cur group. F: H-Cur group. L-Cur, low-dose curcumin, 50 mg/ kg i.p.; M-Cur, middle-dose curcumin, 100 mg/kg i.p.; H-Cur, high-dose curcumin, 150 mg/kg i.p. I: TNF-α. II: IL-1β. Data are presented as the mean ± standard error (n = 5). *P < 0.05 vs. Sham group; #P < 0.05 vs. CLP group; ▲P < 0.05 vs. XBJ group.

Figure 6. Effects of curcumin on survival of rats with CLP-induced liver dys -function. L-Cur, low-dose curcumin, 50 mg/kg i.p.; M-Cur, middle-dose curcum -in, 100 mg/kg i.p.; H-Cur, high-dose curcum-in, 150 mg/kg i.p. The survival rates were observed within 72 h. The results are expressed as cumulative survival, n = 10. The survival rate was estimated by the Kaplan-Meier method and compared by the log-rank test.

Discussion

Sepsis is a disease syn-drome that involves many different aspects of the host immune system. The initial trigger of the disease may be multifaceted, but the most common source is bac-terial infection, especially from the abdominal cavity. Consequently, various ani-mal models of abdominal infection have been devel-oped to mimic the disea- se progression in sepsis,

among which the CLP model

positive rate are higher. The serum cytokine

elevation is relatively flat and longer in dura

-tion. Therefore, CLP animal models have strong

clinical relevance [24, 25]. We thus chose to

perform CLP in the present experiment.

At each time point in this study, animals were

sacrificed by laparotomy. The rats that had undergone CLP exhibited a black, edematous

cecum with purulent exudates in the abdominal

cavity. These findings combined with the histo -logical changes indicated that the septic shock model was successfully established. After treated by XBJ and curcumin, all the parame-ters and histological changes are better than

those of CLP only group, which showed that XBJ

and curcumin were effective.

Liver, as the center of metabolism, is one of the

most important organs of toxins clearance. Numerous studies indicate that liver damage

significantly correlates with the occurrence and

the progression of septic shock. Previous stud-ies suggest that curcumin performs a protec-tive role in relieving hepatic cell damage caused by a variety of factors. This protective effect carried out via the power of increasing superox-ide dismutase (SOD) vitality and inhibiting cyto-chrome C activity [18-20]. Whereas, the effect of curcumin to liver cell in sepsis is still unclear, which is also the aim of the present study. XBJ injection is a traditional Chinese medicine used to treat sepsis and has been approved by

the State Food and Drug Administration of China. The injection combines five traditional Chinese medicines, including Flos Carthami

(the corolla of Carthamus tinctorius L.), Radix

Paeoniae Rubra (the root of Paeonia veitchii

Lynch), Rhizoma Ligustici Chuanxiong (the root

of Ligusticum chuanxiong Hort.), Salviae Miltiorrhizae (the root of Salvia miltiorrhiza

Bge.), and Angelicae Sinensis Radix (the root of

Angelica sinensis (Oliv.) Diels.) [26]. XBJ is wide-ly used in the treatment of sepsis and multiple organ dysfunction syndrome [27-29]. Therefore, we choose the XBJ group as the positive control group to identify the effect of curcumin in sepsis.

PCT, a precursor of calcitonin, is a 116-amino-acid protein secreted in the neuroendocrine cells of the thyroid, lung, and pancreas. PCT can be broken into three distinct molecules with different enzymatic functions: calcitonin

(32 amino acids), katacalcin (21 amino acids), and an N-terminal fragment called aminopro-calcitonin (57 amino acids) [30].

The circulating PCT levels in healthy individuals are very low (usually < 0.1 ng/ml), but rapidly

and significantly increase under conditions of

bacterial infection (> 1000 ng/ml). In sepsis, a rise in the PCT level can be detected at 3 to 4 h, and serial measurements are useful to monitor the response to therapy, embodying good clini-cal acumen [31]. PCT is therefore considered to be a valuable adjunct in the diagnosis and man-agement of sepsis. In the present study, the

PCT level significantly increased in the rats that underwent CLP, whereas it decreased after

treatment with XBJ and curcumin [32]. Therefore, curcumin may effectively control infection, and middle-dose curcumin could work as well as XBJ.

TNF-α is a cytokine with a wide range of biologi -cal effects. It is secreted by macrophages, monocytes, T lymphocytes, and other cells and is synthesized in many tissues and organs

including the heart, liver, and lungs. TNF-α is

mainly secreted by macrophages and plays critical roles in many biological processes, such

as host resistance to infection and the inflam -matory response [33-35].

In one animal experiment, sepsis initiated extensive apoptosis of gastrointestinal epithe-lial and liver cells. This phenomenon then occurred in other organs, resulting in multiple organ failure [36]. Numerous studies have shown that the major promoters of sepsis are

lipopolysaccharides, which finally induce apop

-tosis. TNF-α, as a proinflammatory factor, is the first multifunctional cytokine produced from

lipopolysaccharide-stimulated monocytes and

macrophages [37]. It activates inflammatory cells, which then release IL-1, IL-6, and other inflammatory mediators to extend and expand the inflammatory response, eventually inducing a systemic inflammatory response. IL-1β, a member of the IL-1 cytokine family, is a pleiotro -pic cytokine. It plays a role in the regulation of

systemic and local inflammatory responses

and functions in almost all cells of the human body [38]. Previous studies have suggested

that IL-1β is involved in the apoptosis pathway

physiologi-cal processes such as cell proliferation, differ-entiation, apoptosis, and necrosis [39-41].

TNF-α and IL-1β may therefore elicit the inflam -matory cascade and contribute to the severity of liver injury in sepsis. In the present study,

significantly greater increases in the expres

-sion of TNF-α and IL-1β in the peripheral blood were seen in the CLP group than in the Sham

group, as expected. After treatment with XBJ

and curcumin, the expression of both TNF-α and IL-1β significantly decreased, also as expected. Furthermore, XBJ and M-Cur showed

the best treatment effect.

This study also demonstrated that the protec-tive effect of curcumin varies with the dose used; the middle concentration of curcumin was most effective for septic rat hepatocytes. This might be correlated with the pharmacoki-netics of curcumin. Curcumin is metabolized

mainly by the liver, and the first pass effect is

obvious. After enterohepatic circulation, most ingredients are transformed and cleared [42,

43]. For this reason, the low concentration of

curcumin lacked effective protection. Therefore, in severe sepsis, when large numbers of liver

cells are damaged by inflammatory cytokines

and mediators, a high concentration of curcum-in could curcum-increase the burden of liver metabo-lism. Thus, the middle concentration of curcum-in was most effective for protection of septic rat hepatocytes.

In conclusion, the present study has shown

that curcumin can protect against CLP-induced liver dysfunction by decreasing the TNF-α and IL-1β levels. The pathological examination find

-ings and changes in the PCT level confirmed this conclusion. Furthermore, the present study

found that the protective effect of curcumin may be related to the drug concentration; nei-ther a low nor high dose of curcumin will achieve

the optimal effect. Although we confirmed the beneficial effect of curcumin on hepatocytes in

septic rats, the precise mechanisms remain to be fully elucidated. Clinical trials are also nec-essary to fully realize the potential use of cur-cumin in patients with sepsis.

Disclosure of conflict of interest

None.

Address correspondence to: Dr. Jian-Rui Wei, De- partment of Cardiology, Guangzhou Red Cross

Hospital, Medical College, Jinan University, No. 396 Tongfuzhong Road, Guangzhou 510220, Guandong, PR China. Tel: +8618928900398; E-mail: jianrui_ wei@sina.com

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